Project Summary Diseases arising from lung developmental disorders are widespread and often deadly. Our abilities to develop therapies for these disorders or to engineer artificial lungs in the laboratory are limited by our incomplete understanding of how the complex and stereotyped architecture of the lung develops. The smooth muscle that surrounds the airways (airway smooth muscle, ASM) has recently been shown to play a role in the bifurcation of epithelial buds, an important step in the development of the branched structure of the lung. To help drive bifurcation, ASM must differentiate from the mesenchyme surrounding epithelial bud tips in a precise and asymmetric pattern, but the mechanisms controlling this spatial pattern of differentiation are poorly understood. Signaling molecules, like sonic hedgehog and bone morphogenic protein-4, and mechanical cues, like the pressure inside the developing airway, are known to increase smooth muscle differentiation. I hypothesize that the transpulmonary pressure causes a distribution of mechanical stress in the mesenchyme that specifies the spatial pattern of smooth muscle differentiation around bud tips. Confirming this hypothesis would support a model in which mesenchymal cells around bud tips are primed to be potential ASM precursors by signaling molecules, but the precise pattern of mechanical stress determines which potential precursors differentiate into ASM. To test the hypothesis, I will determine whether stretch can induce airway mesenchymal cells to adopt an ASM phenotype in culture (Aim 1). I will then analyze RNA sequencing data of lungs cultured under high and low pressure to understand potential molecular mechanisms by which transpulmonary pressure is transduced into a morphogenetic signal (Aim 2). Finally, I will use computational models of lung volumetric reconstructions to determine whether bud epithelial geometry and transpulmonary pressure are sufficient to cause stress distributions in the mesenchyme that predict the pattern of ASM differentiation at bud tips (Aim 3). Successfully completing these aims will deepen our understanding of how ASM differentiates and how transpulmonary pressure affects lung development. This knowledge could inform future research into therapies that modulate ASM differentiation to treat congenital lung diseases and future efforts to direct the differentiation of smooth muscle in tissue-engineered lungs.